Objective To investigate the effects of Yiqi Huoxue Lishui Formula (YQHXLSF) on mitochondrial morphology and function in heart failure (HF) rats by activating the AMPK/PGC-1α signaling pathway. Methods HF models were established by left anterior descending coronary artery (LAD) ligation combined with starvation and exhaustive swimming. Postoperatively, rats were randomly divided into sham, model, trimetazidine (TMZ), YQHXLSF medium-dose (M), and high-dose (H) groups. After 4 weeks of treatment, cardiac function was assessed using echocardiography and HE/Masson staining. ROS levels, mitochondrial ultrastructure, and functional changes were detected. Protein expression of the AMPK/PGC-1α pathway was measured by Western blot, while mRNA expression and mitochondrial DNA (mtDNA) replication levels were evaluated using Real-time PCR. Results YQHXLSF significantly improved cardiac function indices, attenuated myocardial fibrosis, reduced ROS levels, enhanced mitochondrial respiratory chain complex activity and membrane potential, and increased ATP content. Western blot and Real-time PCR results demonstrated that YQHXLSF upregulated the expression of p-AMPK, PGC-1α, NRF-1, and TFAM at both protein and mRNA levels, and promoted mtDNA replication. Conclusion YQHXLSF improves cardiac function and myocardial fibrosis, reduces ROS levels, maintains mitochondrial structural and functional stability, promotes mitochondrial biogenesis, and regulates mitochondrial energy metabolism to increase ATP production in HF rats. These effects may be mediated through activation of the AMPK/PGC-1α signaling pathway.